The present invention generally relates to the fields of analytical chemistry and materials characterization. Specially, the present invention relates to fractionation of heterogeneous mixtures of particles on the basis of characteristic frequency dependence of particle polarization and also to analysis of electrochemical properties of particles.
I. 1 Methods of Fractionation and Sorting
Among analytical separation techniques, methods of flow cytometry have been used in sorting of particles (including cells) within a given heterogeneous population. These methods, however, require a dedicated instrument capable of selecting and physically separating from the population those particles which satisfy a given selection criterion. For example, the objective of cell sorting by flow cytometry usually is the selection of those cells within a population displaying a characteristic fluorescence signal.
I. 2 Methods of Particle Analysis
Particle analysis represents a standard procedure of the analytical chemical repertoire that is used to determine physico-chemical and compositional properties [Hunter, xe2x80x9cIntroduction to Modem Colloid Sciencexe2x80x9d, Oxford University Press, Oxford, UK, 1993]. The extensive repertoire of techniques reflects the ubiquitous use of particles of a wide range of sizes, shapes, composition and chemical reactivity in many scientific and industrial applications ranging from medical diagnostics to cosmetics. Characterization of particles is useful in guiding and optimizing production as well as chemical modification particularly of surface properties that determine the stability of particle suspensions and the interaction of particles with molecules in the surrounding medium.
Techniques of the standard repertoire may be grouped as follows: first, sedimentation and centrifugation methods, electroacoustics, light scattering, hydrodynamic methods and dielectric spectroscopy; second, electrical pulse counting, flow cytometry and electrophoretic zeta potential measurements; third, specialized methods including dielectrophoresis (DEP), including its combination with field-flow fractionation methodology.
Methods in the first group apply to bulk suspensions and therefore deliver an average of the measured quantities for a large number of particles. While desirable in situations in which large industrial batches are to be characterized, bulk measurements generally do not lend themselves to miniaturization and are not well suited to the characterization of small numbers of particles, particularly when particle-to-particle variations are of interest or when multiple particle parameters are to be determined simultaneously.
Methods in the second group use a xe2x80x9csingle-filexe2x80x9d serial format of measurement. While these methods determine the properties of individual particles, their implementation, in order to attain sufficiently high processing rates, requires high flow rates of a carrier gas or fluid and high-speed data recording and read-out electronics, generally rendering the equipment complex and expensive. This is so especially when multiple parameters are to be determined for each particle. For example, multi-color detection in flow cytometers can require the use of multiple lasers and multiple photomultiplier tubes. The determination of the zeta potential along with particle size in state-of-the-art equipment requires the measurement of electrophoretic mobility of particles traveling along a narrow capillary in response to a DC voltage applied along the channel as well as the application of light scattering with associated light source and read-out, as in the ZetaSizer (Malvern Instruments, Southborough, Mass.). Miniaturization, as in the case of flow cytometry [Fu, A. N., Spence, C., Scherer, A., Arnold, F. H. and Quake, S. R. A Microfabricated Fluorescent-Activated Cell Sorter, Nature Biotechnology, Vol. 17, November 1999, 1109-1111.], retains the serial mode of processing and thus encounters the constraint of limited throughput.
Methods in the third group include various dielectrophoretic techniques to characterize, classify and fractionate low conductivity particle suspensions [Becker, F. F., Gascoyne, P. R. C., Huang, Y. and Wang, X -B, Method and Apparatus for Fractionation Using Generalized Dielectrophoresis and Field Flow Fractionation, U.S. Pat. No. 5,888,370, Mar. 30, 1999; Rousselet, J., Markx, G. H. and Pethig, R., Separation of Erythrocytes and Latex Beads by Dielectrophoretic Levitation and Hyperlayer Field-Flow Fractionation, Colloids and Surfaces A: Physicochemical and Engineering Aspects, 140 (1998) 209-216; Pethig, R., Markx, G. H., Apparatus for Separating by Dielectrophoresis, U.S. Pat. No. 5,814,200, Sep. 29, 1998; Pohl, H. A., Continuous Dielectrophoretic Cell Classification Method, U.S. Pat. No. 4,326,934, Apr. 27, 1982; Parton, A., Huang, Y., Wang, X-B., Pethig, R., MacGregor, A. R., and Pollard-Knight, D. V., Methods of Analysis/Separation, U.S. Pat. No. 5,653,859, Aug. 5, 1997; Crane, S., Dielectrophoretic Cell Stream Sorter, U.S. Pat. No. 5,489,506, Feb. 6, 1996; Benecke, W., Wagner, B., Hagedorn, R., Fuhr, G., Muller, T., Method of Continuously Separating Mixtures of Microscopic Dielectric Particles and Apparatus for carrying through this method, U.S. Pat. No. 5,454,472, Oct. 3, 1995]. Various publications, which are cited throughout the application, are hereby incorporated by reference in their entirety.
Coupled with a suitable imaging system, techniques within this group can provide single particle information. The particle classification is achieved by exposing the particle mixture to a non-uniform AC electric field which is generated by applying an AC voltage to multiple sets of planar patterned microelectrodes. Such a scheme in general requires a complex electrode signaling/addressing scheme.
Also, a continuous mode of operation requires, in the simplest case, coupling to an external flow or the use of field flow fractionation. Simple dielectrophoretic setups do not generally allow for quantitative evaluation for the surface potential or surface conductivity of particles. While these quantities maybe determined from electrorotation spectra of single particles, this approach requires a complex and time-consuming set-up to confine a single particle at the origin of a rotating electric field whose frequency is scanned while recording the rotational motion of the confined particle. Aside from the complexity of the equipment required to implement the requisite experimental configuration, the low throughput of the method presents a serious disadvantage when averages of multiple particles are desired.
The present invention provides a method and apparatus for the fractionation of a mixture of particles and for the analysis of particles, based at least in part on the technology designated xe2x80x9cLEAPSxe2x80x9d, described in PCT International Application No. WO 97/40385 to Seul. The present invention uses LEAPS (referring to xe2x80x9cLight-Controlled Electrokinetic Assembly of Particles near Surfacesxe2x80x9d) to fractionate a heterogeneous mixture of particles on the basis of, e.g.: morphological properties including size and shape; surface composition relating to certain electrochemical properties, including surface potential and surface conductivity; and specific and non-specific adhesion to exposed surfaces provided within the apparatus. The present invention also provides for the use of LEAPS to determine various physical or chemical properties of particles, including morphological properties, surface electrochemical properties and surface chemical properties such as the particles adhesiveness for given surfaces of the apparatus or for other particles.
The present invention provides a method and apparatus for fractionation of particle mixtures on the basis of the respective characteristic relaxation frequencies of the constituent particles or particle populations. In certain embodiments of such method, a plurality of particles are suspended at an interface between an electrolyte solution and a light-sensitive electrode. The particles comprise at least two types, each type having a distinguishable relaxation frequency. An electric field, having a frequency that is less than or equal to the relaxation frequency of at least one of said particle types but greater than the relaxation frequencies of other particle types, is generated at the interface by application of an AC voltage. In addition, the interface is illuminated with a predetermined light pattern. The illumination in combination with the electric field produces fractionation of the particles having relaxation frequencies greater than or equal to the frequency of the electric field from other particles, e.g., by assembly of particles having a relaxation frequency greater than the frequency of the applied electric resulting in formation of a planar array of substantially one layer of particles in an area on the electrode designated by the pattern of illumination. In certain embodiments, the illumination affects the electrolyte-electrode interfacial impedance.
In certain preferred embodiments, the electrode comprises a silicon electrode which is coated with a dielectric layer. In certain preferred embodiments, an additional electrode is provided such that the light-sensitive electrode and the additional electrode are substantially planar and parallel to one another and separated by a gap (e.g., in a sandwich configuration), with the electrolyte solution containing the particles being located in the gap.
In certain other embodiments of the present invention, patterned electrode is used instead of or in combination with the illumination to fractionate a mixture of particles. In one such example, a first electrode positioned in the first plane and a second electrode positioned in the second plane different from the first plane is provided with a gap between the electrodes, an arrangement herein referred to as a sandwich configuration. A mixture of particles is suspended in an electrolyte solution and located in the gap. The second electrode comprises a planar electrode having a surface and an interior, either of which maybe patterned to modify the spatial distribution of the interfacial (interface between the electrolyte solution and the second electrode) electric field. When an electric field is generated between the first and the second electrode by applying an AC voltage between the two, the electric field in combination with the patterning of the electrode produces fractionation of the particles according to relaxation frequency; for example, particles having a relaxation frequency greater than the frequency of the applied electric field move toward designated regions of the second electrode surface as provided by illumination or patterning and therein form a planar array of substantially one layer. In preferred embodiments, the second electrode maybe patterned by spatially modulated oxide growth, surface chemical patterning or surface profiling; preferably, the patterning affects the impedance of the electrolyte solution and second electrode interface. In preferred embodiments, the second electrode comprises a silicon electrode which is coated with a dielectric layer.
In certain embodiments, the difference in relaxation frequency of particles within a heterogeneous mixture corresponds to a difference in particle size or surface composition, thus permitting fractionation of the mixture into homogeneous subpopulations of particles according to size or surface composition, respectively. The process of fractionation may also be monitored using a video detector or camera.
Accordingly, the present invention provides a method and apparatus for fractionation of particle mixtures based on the characteristic relaxation frequencies of said mixture""s constituent particles or particle subpopulations. The present invention provides for the determination of the frequency dependence of the electrohydrodynamic forces that govern particle transport into designated low-impedance areas of the electrode and mediate formation of particle arrays in those designated areas. As described herein, a particle relaxation frequency, xcfx89R, is a property that is associated with the particle-solution interface and may be determined from the condition that array assembly requires xcfx89xe2x89xa6xcfx89R,: particles not meeting this criterion will be excluded from low-impedance areas.
As used herein, the term xe2x80x9cfractionationxe2x80x9d refers to separation or sorting of particles, including separation of multi-component particle mixtures into their constituent subpopulations. Particles may be separated on the basis of the characteristic frequency dependence of the particle polarization, said dependence reflecting physical or chemical properties of the particles.
The present invention also provides for a method and apparatus for the analysis of particles in order to determine various physical or chemical properties of said particles, including size, morphology, surface potential and surface conductivity. For example, the relaxation frequency and/or maximal velocity of transport of the particles are used for particle characterization.
In certain embodiments of the present invention, a plurality of particles suspended in an interface between an electrolyte solution and a light-sensitive or a patterned electrode are provided, as described above in connection with particle fractionation. When an electric field is generated at the interface by the application of an AC voltage, the frequency of the electric field is adjusted to produce formation of a planar array of substantially one layer of particles in an area designated by the pattern of illumination or electrode patterning. From the analysis of particle transport in the course of array assembly, the relaxation frequency and the maximal velocity of transport of the particles may be determined. Alternatively, the particle relaxation frequency also maybe determined by analyzing the response of the array configuration to changes in the frequency of the applied electric field.
The present invention also provides for the determination of the frequency-dependent and voltage-dependent maximal velocity, vmax attained by particles crossing impedance gradients in the course of array assembly. As described herein, vmax may be determined by image analysis and particle tracking. In certain embodiments, the combination of xcfx89R and vmax provides for the simultaneous determination of the particles"" surface (xe2x80x9czetaxe2x80x9d) potential, xc3x8s, and surface conductivity, "sgr", e.g., based on numerical analysis of phenomenological equations of particle motion described herein.
Once these values are found, they may be used to characterize the particles, e.g., determine the zeta potential of the particles and/or the mobility of ions or molecules within the electrolyte solution in proximity to the particle surface. In preferred embodiments, the relaxation frequency and the maximal velocity are used together in particle characterization. In certain preferred embodiments, the present invention permits the simultaneous determination of multiple particle properties, such as zeta potential and ionic or molecular mobility. In other embodiments, the present invention permits the determination of one particle property of interest independently from the determination of others. For example, in certain embodiments, the ionic or molecular mobility associated with the particle-solution interface may be determined from the relaxation frequency alone.
The term xe2x80x9cparticles,xe2x80x9d as used herein include colloidal beads, eukaryotic and procaryotic cells, micelles, vesicles, and emulsion droplets. In preferred embodiments, the particles comprise colloidal beads, or eukaryotic or procaryotic cells.
In contrast to prior art methods of fractionation of particle mixtures such as field flow fractionation, the methods of the present invention provide a parallel format based on the manipulation of a multiplicity of individually imaged particles in a simple sandwich geometry which provides for longitudinal and transverse electrohydrodynamic forces to mediate fractionation on the basis of the frequency-dependent polarization of the particles of interest. Specifically, the methods of the present invention provide for the fractionation of one subpopulation of particles at a time from the remainder.
In contrast to prior art methods of sorting such as fluorescence-activated particle and cell sorting, the methods of the present invention provide for a parallel process accommodating a multiplicity of particles thereby enhancing processing speed. The methods of present invention also are well suited for miniaturization.
In contrast to prior art methods of particle analysis which are applied to bulk particle suspensions and yield ensemble averages of the particle properties of interest, the methods and apparatus of the present invention provide for the miniaturization of the processes of analysis. In contrast to prior art serial methods of analysis of particle mobility, the methods of present invention invoke a collective phenomenon of particle array assembly as the basis for a parallel mode of simultaneous analysis of multiple particle properties accessible either to imaging and image analysis or to light scattering. The methods of the present invention provide for the simultaneous, yet independent determination of morphological parameters including size, orientation and polydispersity, as well as for electrochemical parameters including surface potential and surface conductance.
In contrast to these prior art methods, the on-chip implementation of the fractionation methods of the present invention are readily suitable for miniaturization and integration with additional preparative and analytical procedures in an integrated on-chip analytical environment.